The present invention is directed to D-alanine racemase mutants of mycobacterial species. The D-alanine racemase gene (alrA) is involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the bacterial cell wall. The present invention is also directed to methods of making live-attenuated vaccines against pathogenic mycobacteria using such alrA mutants and to the vaccines made according to such methods. The present invention is further directed to use of alrA mutants in methods for screening antimycobacterial agents that are synergistic with peptidoglycan inhibitors. Finally, the present invention is directed to methods to identify new pathways of D-alanine biosynthesis for use in developing new drugs targeting peptidoglycan biosynthesis in mycobacteria and to identify vaccines useful against pathogenic mycobacteria.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Mycobacteria cause a number of diseases in humans and animals including tuberculosis, which is the leading cause of human death from an infectious disease in the world (Bloom & Murray, 1992). M. tuberculosis is the principal cause of tuberculosis in humans and other primates and is occasionally seen in dogs, pigs and cattle (O'Reilly & Daborn, 1995). In contrast, M. bovis, the etiologic agent of bovine tuberculosis, has a wide host range and infects ruminants, carnivores, and primates, including humans (Pritchard, 1988). For M. bovis and M. tuberculosis in both humans and animals, contaminated aerosols are the most common routes of transmission (Carleton, 1993). Other mycobacterial pathogens of importance are M. avium, M. paratuberculosis, and M. leprae. M. avium is the agent of tuberculosis in birds but its major significance is as an opportunistic pathogen of AIDS patients (Inderlied et al., 1993). M. paratuberculosis is the etiologic agent of Johne's disease, a granulomatous enteritis in ruminants and it has also been linked to a potential etiology of a type of inflammatory bowel disease (Crohn's disease) in humans (Cocito et al., 1994; Harris and Barletta, 2001). Finally, M. leprae infects humans and armadillos. Though leprosy in humans has low mortality, its morbidity is quite high in affected areas, estimated to have been 10-12 million in the 1980s (Noordeen, 1991). All these diseases caused by mycobacteria are characterized pathologically by the formation of granulomatous nodules (tumor-like masses caused by chronic inflammatory processes) or “tubercules” that are seen in advanced cases. Due to the significance of mycobacterial diseases, prevention and control measures, including vaccines, diagnostics and therapies are of major importance.
Microorganisms of the M. avium complex have achieved prominence as major opportunistic pathogens of AIDS patients. M. avium is naturally resistant to most firstline antituberculosis drugs (Inderlied et al., 1993). This threat to public health has been partially met by therapy with appropriate antimicrobial agents, but unfortunately drug-resistant M. avium and M. tuberculosis strains readily appear (Chaisson et al., 1994; Espinal et al., 2000), underscoring the need to develop new and more effective anti-mycobacterial agents.
Mycobacterium smegmatis is a fast-growing nonpathogenic mycobacterial species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. The D-alanine racemase gene (alrA) is involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. Biosynthesis of the mycobacterial cell wall has received considerable attention in the search for inhibitors useful for drug therapy (Chatterjee, 1997). These cell walls display a complex architecture of glycolipids and proteins linked to the mycolyl-arabinogalactan-peptidoglycan backbone (McNeil and Brennan, 1991). This structure is a barrier that contributes to drug resistance (Trias and Benz, 1994), and many of its components have been found to play a major role in pathogenesis (Daffe and Draper, 1998). The analysis of the M. tuberculosis genome sequence suggests that peptidoglycan biosynthesis in mycobacteria follows the general pathway of other bacteria, including the formation of the basic building block D-alanyl-D-alanine (Belanger and Inamine, 2000; Cole et al., 1998). D-alanine racemase (Alr) catalyzes the conversion of L-alanine into D-alanine (Julius et al., 1970), and D-alanine-D-alanine ligase catalyzes the subsequent dimerization of D-alanine into the key dipeptide D-alanyl-D-alanine (Neuhaus, 1962). The corresponding enzymes from both Escherichia coli (Lambert and Neuhaus, 1972; Neuhaus and Lynch, 1964) and mycobacteria (Cáceres, 1999; David et al., 1969) are inhibited by D-cycloserine (DCS), a D-alanine analog (Neuhaus, 1967). The dipeptide is then added to the UDP-tripeptide precursor by the action of the D-alanine-D-alanine adding enzyme that completes the reactions of the D-alanine branch of peptidoglycan assembly (Walsh, 1989).
DCS is particularly effective against mycobacteria albeit with marked side effects (Cummings et al., 1955; Yew et al., 1993). Moreover, overproduction of Air in Mycobacterium smegmatis, Mycobacterium intracellulare, and Mycobacterium bovis BCG leads to a DCS-resistant phenotype. The M. smegmatis enzyme is inhibited by DCS in a concentration-dependent manner (Cáceres et al., 1997). Likewise, the M. avium and M. tuberculosis enzymes produced from E. coli recombinant clones are also inhibited by DOS (Strych et al., 2001). Nonetheless, the specific characteristics of the mycobacterial enzymes involved in peptidoglycan biosynthesis, including the essentiality of each of their functions, remain unknown. Such knowledge would be important to the design of specific inhibitors that would serve as novel bactericidal agents to treat M. tuberculosis and M. avium infections. Furthermore, the inactivation of the genes encoding for these enzymes may lead to the generation of attenuated strains of pathogenic mycobacteria that could serve as candidate vaccines against tuberculosis. 
M. smegmatis has been extensively used as a model system for M. tuberculosis and other mycobacteria. M. smegmatis is nonpathogenic, requiring less stringent containment facilities, and it grows at a relatively high rate in a variety of defined and nutrient-restricted media (Jacobs, 2000). M. smegmatis has been used to study drug resistance mechanisms (Cáceres et al., 1997; Peteroy et al., 2000; Telenti et al., 1997) and basic physiological processes including the synthesis of peptidoglycan precursors (Cirillo et al., 1998; Pavelka and Jacobs, 1996). Insights gained from these studies can then be applied to the pathogenic mycobacteria.